Acta Biomaterialia 3 (2007) 23–31

www.actamat-journals.com

Covalently crosslinked chitosan hydrogel: Properties ofin vitro degradation and chondrocyte encapsulation

Yi Hong a, Haiqing Song b, Yihong Gong a, Zhengwei Mao a,Changyou Gao a,*, Jiacong Shen a

a Department of Polymer Science and Engineering, Zhejiang University, Key Laboratory of Macromolecule Synthesis and Functionalization,

Ministry of Education, Hangzhou 310027, Chinab The College of Management, Zhejiang University, Hangzhou 310027, China

Received 16 February 2006; received in revised form 15 June 2006; accepted 27 June 2006

Abstract

In vitro degradation and chondrocyte-encapsulation of chitosan hydrogel made of crosslinkable and water-soluble chitosan derivative(CML) at neutral pH and body temperature were studied with respect to weight loss, cytoviability, DNA content and cell morphology. In

vitro degradation of the chitosan hydrogels was sensitive to their crosslinking degree and existence of lysozyme in the solution. Chitosanhydrogel (Gel-I5) fabricated from 1% CML and 5 mM ammonium persulfate (APS)/N,N,N 0,N 0-tetramethylethylenediamine (TMEDA)displayed no degradation in phosphate buffered saline (PBS) after 18 d, but degraded completely at 8 d in 1 mg/ml lysozyme/PBS. Thechitosan hydrogel fabricated from 10 mM APS/TMEDA was non-degradable even in lysozyme/PBS solution after 18 d. The hydrogelloaded with chondrocytes in cell culture medium, however, was susceptible to degradation during the in vitro culture. In vitro culture ofthe encapsulated chondrocytes in the chitosan hydrogel demonstrated that the cells retained round shaped morphology and could survivethrough a 12 d-culture period, although the DNA assay detected an overall reduction of the cell number. These features provide a greatopportunity to use the chitosan hydrogel as an injectable scaffold in tissue engineering and orthopaedics.� 2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Chitosan; Hydrogel; Degradation; Chondrocytes; Injectable scaffold

1. Introduction

Injectable scaffolds are promising substrates for tissueengineering because of the in vivo culture environment,minimal invasion and low cost, and so on [1,2]. Variousmaterials including microspheres and hydrogels have beenemployed as injectable scaffolds. Hydrogels have a similarmicrostructure to the extracellular matrix (ECM) and canundergo a sol–gel transition in very mild conditions. Thehydrogel precursor loaded with targeted cells can beinjected into the damaged site and experiences a gel transi-tion in situ due to physical or chemical stimuli. The encap-

1742-7061/$ - see front matter � 2006 Acta Materialia Inc. Published by Else

doi:10.1016/j.actbio.2006.06.007

* Corresponding author. Tel.: +86 571 87951108; fax: +86 57187951948.

E-mail address: cygao@mail.hz.zj.cn (C. Gao).

sulated cells grow within the hydrogel and secrete their ownECM to reestablish the damaged tissue.

The injectable hydrogels can be originated from eithernatural biomacromolecules such as collagen [3,4], chitosan[5,6], alginate [7–9] and hyaluronan [10,11], or syntheticmaterials such as poly(ethylene glycol) (PEG) [12,13],poly(propyl fumarate) (PPF) [14,15] and Pluronic F127[16]. The in vivo gelation of these materials can be triggeredby temperature [17], thermal chemical crosslinking [18,19],photo crosslinking [20,21], ionic crosslinking [22] and self-assembly [23]. Among these gelation methods, thermalchemical crosslinking is relatively easy without limitationof the injection depth. The biggest problem of the potentialcytotoxicity caused by the initiators and chemical reactioncan be resolved by optimizing gelation conditions and thetype of initiators used.

vier Ltd. All rights reserved.

24 Y. Hong et al. / Acta Biomaterialia 3 (2007) 23–31

Chitosan has been widely applied in drug delivery, genecarriers and tissue engineering because of its biocompati-bility and biodegradability [24,25]. However, unmodifiedchitosan can be only dissolved in acidic solution becauseof its strong intermolecular hydrogen bonds. This limitsits applications, especially as an injectable scaffold. Theaddition of glycerol-2-phosphate (b-GP) can adjust thechitosan solution from acidic to neutral, thus the chitosanhydrogel can be formed at neutral pH and body temper-ature [5]. Injection and cultivation of this hydrogel loadedwith chondrocytes in a mouse has formed proteoglycan-rich matrix in vivo. Moreover, mesenchymal stem cells(MSCs) were encapsulated in an injectable thermosensi-tive chitosan hydrogel (water-soluble chitosan-g-poly-(N-isopropylacrylamide)) [26]. In vivo culture showed thatthe MSCs could differentiate to chondrocytes, and toform cartilage after the cell–hydrogel construct was injec-ted into the submucosal layer of the bladder of a rabbitand cultured for 14 weeks. Recently, chitosan moleculeswere also grafted with N-isobutyryl groups to obtain athermosensitive chitosan hydrogel [27], or vanillin orhydroxybenzaldehydes to obtain an ultraviolet (UV) cros-slinkable chitosan hydrogel with better biocompatibility[28].

In our previous work [29], a water-soluble and cros-slinkable chitosan derivative (CML, see Scheme 1a) havingC@C double bonds in its molecules was synthesized bysequentially grafting of methacrylic acid (MA) and lacticacid (LA) via the reaction between amino groups andcarboxyl groups under the catalysis of carbodiimide. TheCML is readily soluble in pure water and does not precip-itate till pH 9. At neutral pH and body temperature, the

Scheme 1. (a) Molecular structure of water-soluble and crosslinkable chitosan dlactic acid (LA) onto the pendant amine groups. The structure does not represenhydrogel network linked by alkyl chains which are formed via C@C polymeri

three-dimensionally crosslinked chitosan hydrogel (Scheme1b) is then formed by polymerization of the C@C doublebonds under the initiation of a redox system, ammoniumpersulfate (APS)/N,N,N 0,N 0-tetramethylethylenediamine(TMEDA). Analyzed by in vitro cytotoxicity assay andin vivo implantation, the hydrogel fabricated from 1%CML and 5 mM APS/TMEDA has shown minimal ornegligible cytotoxicity and is histocompatible; that is, notissue necrosis and malignant infection were evidenced,although acute inflammation was observed in the initialstage [29]. In this work, we shall report the in vitro degra-dation behavior and the encapsulation performance forchondrocytes. The growth behaviors of the encapsulatedchondrocytes will also be evaluated. These properties areindispensable if the hydrogel is to be used as an injectablescaffold.

2. Experiments

2.1. Materials

Chitosan (>95%, average Mg = 620,000 and deacetyla-tion degree (tested via elemental analysis) = 78%) wasobtained from Haidebei Company, Jinan, China. Metha-crylic acid (MA, AR, Shanghai Chemical Reagent Company,China) and ammonium persulfate (APS, >98%, Yixing Sec-ond Chemical Reagent Company, China) were purified viadistillation under reduced pressure and recrystallization,respectively. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDAC) was purchased from Sigma. Lacticacid (LA, 85–90%, Shuanglin Chemical Reagent Company,Zhejiang, China) and N,N,N0,N0-tetramethylethylenediamine

erivative (CML), which is obtained by grafting methacrylic acid (MA) andt the real ratio between each monomer unit. (b) Illustration of the chitosan

zation.

Y. Hong et al. / Acta Biomaterialia 3 (2007) 23–31 25

(TMEDA, >98%, Qianjin Chemical Reagent Factory,Shanghai, China) were used directly without purification.

2.2. Synthesis of water-soluble and crosslinkable chitosan

derivative (CML)

The synthesis of CML has been reported previously bygrafting MA and LA onto chitosan chain via condensationbetween the amine groups and the carboxyl groups underthe catalysis of carbodiimide [29]. Briefly, after 800 mgchitosan (CS) was dissolved in 100 ml water containing420 ll MA, 930 mg EDAC was added. The reaction tookplace over 24 h at room temperature under agitation. Inorder to remove the free MA and other byproducts, themixture was dialyzed in a filter membrane with a molecularweight cut off of 10,000 Da against a large amount oftriple-distilled water for 3 d. Finally, MA grafted chitosan(CM) was obtained by freeze-drying. Subsequently, theCM was dissolved in 100 ml water containing 420 ll LAovernight, and then 930 mg EDAC was added. The mix-ture was stirred at room temperature for 24 h. After thesame purification procedures as described above, thewater–soluble and crosslinkable chitosan derivative(CML) was obtained. The grafting ratios of MA and LAwere quantified as 23% and 52% via elemental analysis,respectively.

2.3. Fabrication of chitosan hydrogel

CML aqueous solution was gelled by radical polymeri-zation under the initiation of a redox system including oxi-dant APS and reducer TMEDA. APS and TMEDA werepreviously both made into 1 M solutions. 1% CML aque-ous solution was mixed with APS solution, followed bythe same amount of TMEDA solution. Then the mixturewas injected into a mold by a syringe. The chitosan hydro-gel was formed in the mold at a temperature of 37 �C. Thefinal concentration of both APS and TMEDA in this mix-ture was controlled at 5 or 10 mM, respectively. Gel-I5 andGel-I10 represent chitosan hydrogels formed at initiatorconcentrations of 5 and 10 mM, respectively.

2.4. In vitro degradation of the chitosan hydrogel

0.5 ml 1% CML solution with 5 or 10 mM initiatorwas placed in a cylindrical mold for 24 h at 37 �C toobtain the chitosan hydrogel. After reaching equilibriumin PBS at 37 �C for 24 h, the hydrogels were weighed(W0). After being lyophilized, they were weighed again(W1). Then the hydrogels were placed into phosphatebuffered saline (PBS) or 1 mg/ml lysozyme/PBS solutionat 37 �C. The hydrogels were taken out and weighed(W2) every 1 d or 3 d, then were freeze-dried and weighedagain (W3). The wet weight remaining ratio (with water)and the dry weight remaining ratio (without water) werecalculated as follows:

Wet weight remaining ratio ð%Þ ¼ W 2=W 0 � 100% ð1ÞDry weight remaining ratio ð%Þ ¼ W 3=W 1 � 100% ð2Þ

2.5. Chondrocyte culture

Chondrocytes were isolated from the cartilage tissue ofrabbit ears (Japanese big ear white). The rabbits weresacrificed under the institutional ethical guidelines.Briefly, cartilage tissue was cut into small pieces. Chon-drocytes were isolated by incubating the cartilage piecesin Dulbecco’s minimum essential medium (DMEM) con-taining 0.2% collagenase type II (Sigma) at 37 �C for 6 hunder agitation. The isolated chondrocytes were centri-fuged, resuspended in DMEM supplemented with 10%fetal bovine serum (FBS), 100 U/ml penicillin and100 lg/ml streptomycin. The cell suspension was thenseeded in an 11 cm plastic tissue culture dish (Falcon)and incubated in a humidified atmosphere of 95% airand 5% CO2 at 37 �C. After a confluent cell layer wasformed, the cells were detached using 0.25% trypsin inPBS and were resuspended in PBS, and used for theexperiments.

2.6. Chondrocyte encapsulation in the chitosan hydrogel

100 mg CML was sterilized under UV radiation for 3 hand then dissolved in 10 ml sterilized PBS to obtain a 1%CML/PBS solution. APS and TMEDA were made into1 M PBS solutions and sterilized via filtering through a fil-ter with pore size of 0.22 lm. 50 ll 1 M APS/PBS solutionand 50 ll 1 M TMEDA/PBS solution were sequentiallyadded into the CML/PBS solution. The final initiator con-centration was 5 mM. Then, 500 ll cells/PBS suspensionwas mixed with the CML/initiator solution with gentle agi-tation. The final cell density in the hydrogel precursor was4 · 106/ml. 250 ll cells/hydrogel precursor was injectedinto a mold using a 1 ml syringe. After being held at37 �C for �8 min in an incubator, the cells/hydrogel con-struct was formed. Then 1 ml DMEM supplemented with10% FBS was added. After 30 min, the cells/hydrogel con-struct was transferred to a well of 24-well culture plate, and2 ml DMEM supplemented with 20% FBS was added.Over the next 3 h, the culture medium was changed threetimes to remove the uncrosslinked hydrogel and other sol-uble substances. During the 12-d culture period, the culturemedium was changed every 3 d.

2.7. In vitro degradation of the chitosan hydrogel containing

chondrocytes

The cells/hydrogel construct incubated in DMEM sup-plemented with 20% FBS were weighed every 3 d. Afterbeing freeze-dried, the cells/hydrogel construct wasweighed again. Pure chitosan hydrogel without cells wasused as control and weight loss was measured at the sameconditions.

26 Y. Hong et al. / Acta Biomaterialia 3 (2007) 23–31

2.8. MTT assay

After injection of 100 ll MTT (3-(4,5-dimethyl) thiazol-2-yl-2,5-dimethyl tetrazolium bromide, 5 mg/ml) into thecells/hydrogel construct, the construct was continuouslycultured for another 4 h. During this period, viable cellscould reduce the MTT to formazan pigment, which wasdissolved by 1 ml dimethyl sulphoxide (DMSO) afterremoval of the culture medium. The hydrogel was smashedand centrifuged at 11,000 rpm to ensure the completeextraction of the formazan pigment by DMSO. The absor-bance at 570 nm was recorded under a microplate reader(Bio-Rad 550).

2.9. DNA assay

At each time interval the cells/hydrogel construct wastaken out from the 24-well culture plate and were storedat �40 �C in a 2 ml plastic tube. After the experimentwas finished, all samples were frozen and thawed. Thisprocess was repeated several times. 1 ml 10 mg/mlpapain/EDTA (disodium ethylenediaminetetraacetic acid)solution was added and samples were placed in a waterbath at 65 �C overnight to ensure the complete extractionof DNA. 100 ll solution was mixed with 2 ml 1 lg/mlbisBENZIMIDE (Hoechst 33258, Sigma)/FluorescentAssay Buffer solution and the fluorescent intensity of thismixture was measured rapidly on a fluorescence spectro-meter (RF-5301PC, Shimadzu). The excitation wave-length was 360 nm and the emission wavelength was460 nm [30].

2.10. Physical characterization

After freeze-drying, the degraded chitosan hydrogelswere coated with gold and then their structures wereobserved by scanning electron microscopy (SEM, SIRION,FEI). Chondrocytes encapsulated within the hydrogelswere stained by 20 lg/ml fluorescein diacetate (FDA)/40 lg/ml ethidium bromide (EB) and observed by confocallaser scanning microscopy (CLSM, Bio-Rad Radiance

Fig. 1. Weight remaining ratio of chitosan hydrogel (Gel-I5) in PBS and 1 mg/ratio; (b) dry weight remaining ratio.

2100). FDA and EB were used to stain the viable and thedead cells, respectively. Chondrocyte morphology wasobserved by SEM (Stereoscan 260, Cambridge) after fixa-tion, sequential dehydration, critical point drying and goldcoating.

2.11. Statistical analysis

Data from all studies were analyzed using ANOVA.Results are reported as mean ± standard deviation. Thesignificant level was set as p < 0.05.

3. Results and discussion

3.1. In vitro degradation

Chitosan is degradable in vitro at a slow rate. In thepresence of lysozyme, however, the degradation speedcan be accelerated [31]. To mimic the in vivo degradationperformance, lysozyme was added and the results werecompared with those in PBS (Fig. 1). In PBS, the wetweight remaining ratio of Gel-I5 decreased from 100% toabout 85%, while the dry hydrogel weight remaining ratiohad no significant difference (p > 0.05) during the 18 d-degradation (Fig. 1). In 1 mg/ml lysozyme/PBS solution,the wet weight remaining ratio of Gel-I5 remainedunchanged during the first 2 d, and then increased abnor-mally to 128% at the 4th day. Subsequently the wet weightremaining ratio decreased linearly and rapidly (Fig. 1a) till8 d, at which time the chitosan hydrogel disappeared com-pletely. The dry weight remaining ratio showed a similartendency to change as shown in Fig. 1b, except there wasno significant difference during the first 3 d. Macroscopi-cally, the shape of Gel-I5 was reasonably well maintainedin PBS till 18 d, but lost completely in lysozyme/PBS solu-tion at 8 d. One can thus conclude that the chitosan hydro-gel is hardly degradable in PBS, but can be degradedrapidly in lysozyme/PBS solution. Since no significant var-iation of the dry weight was recorded (Fig. 1b), the wetweight loss of the Gel-I5 in PBS should be attributed tothe dehydrating effect of the hydrogel.

ml lysozyme/PBS at 37 �C as a function of time: (a) wet weight remaining

Y. Hong et al. / Acta Biomaterialia 3 (2007) 23–31 27

The weight increase of the wet hydrogel in lysozymesolution is the result of partial degradation, which endowsthe hydrogel with a larger degree of swelling and theabsorption of a greater amount of water. The connectivityof the chitosan hydrogel network was maintained by thecrosslinking of C@C double bonds, which yields alkyl link-ages (shown in Scheme 1b). Therefore, degradation of thehydrogel is caused by the breakage of the glycosidic bondsof the chitosan molecules and the amide bonds of graftedMA. During the initial stage of degradation, breakage ofa small number of glycosidic bonds and amide bonds can-not damage the whole hydrogel network, but the lattice sizeof the networks will enlarge. As a result, the balance swell-ing ratio increases. When the breakage of the glycosidicbonds and the amide bonds reaches a critical value, thewhole crosslinking network will be disjointed, resulting indisappearance of the hydrogel.

When the concentration of the initiator was doubled,the resulting hydrogel (Gel-I10) showed very strong abilityagainst lysozyme degradation (Fig. 2). The wet hydrogelweight decreased slowly with prolongation of degradationtime. At 18 d, the hydrogel still remained at 66% of itswet weight. The dry hydrogel weight, however, had notshown significant change during this 18 d-degradation(p > 0.05). This would mean that the Gel-I10 cannot bedegraded even in lysozyme/PBS solution during 18 ds.Again, the apparent wet weight loss is attributed to thedehydration effect. It is understandable that a higher con-centration of initiator will form more alkyl linkage, andthen yield a higher crosslinking degree. This is confirmedby the fact that the swelling ratio of Gel-I10(20.91 ± 4.42 times) in water at 37 �C is much smaller thanthat of the Gel-I5 (31.15 ± 3.80 times) [29]. Polymerizationof the chitosan macromonomers is largely dependent onthe probability of encountering the C@C double bonds.Crosslinking can take place only if a macromolecular rad-ical is close enough to another C@C bond. The macro-molecular chains have very low moving ability and areconfined within a limited spatial volume. A higher concen-tration of initiator will create more macromolecular radi-cals at a definite volume. As a result, there will be a

Fig. 2. Weight remaining ratio of chitosan hydrogel (Gel-I10) in 1 mg/mllysozyme/PBS at 37 �C as a function of time.

higher chance for the macromolecular radicals to reactwith other C@C bonds. The rather high crosslinking maybring about stereo-hindrance of the enzyme for approach-ing the glycosidic bonds and amide bonds, also leading tothe difficulty of degradation even in lysozyme solution.

The change in the structure of the chitosan hydrogelsduring the degradation was observed by SEM (Fig. 3).Although the structure in the dry state cannot fully repre-sent the real situation of the hydrogel in the wet state, theseimages can still convey some hints of the degradation effect.The white regions reflect the existence of the PBS salt crys-tals. There were many larger and irregular pores throughoutthe lyophilized hydrogels with smooth pore walls regardlessof the degradation time of Gel-I5 in PBS (Fig. 3a–c). All thepores are interconnected through all the dry hydrogel. Thisis understandable since the solid content (chitosan content)is very low (1%) and freeze-drying generally creates a scaf-fold with open pore morphology. The structures of theGel-I10 in lysozyme/PBS (Fig. 3g–i) kept unchanged andwere similar to those of Gel-I5 in PBS from 1 d to 18 d.However, the pores of the Gel-I5 in lysozyme/PBS col-lapsed together, with many rumples on the coarse porewalls at 4 d (Fig. 3e) and 7 d (Fig. 3f), although the structureat 1 d (Fig. 3d) was still similar to that of Gel-I5 in PBS(Fig. 3a). The porous structure basically disappeared, espe-cially after degradation for a longer time (Fig. 3f). Thedegradation weakens the mechanical strength more or less,leading to a larger extent of collapse of the hydrogel upondrying. These observations are consistent with the resultsshown in Figs. 1 and 2.

3.2. In vitro degradation with the encapsulated chondrocytes

The degradation performance of the hydrogel in theexistence of cells is crucial for practical application. Firstly,the gelation time (from the mixing to the state that the mix-ture loses its flow ability [29]) in PBS was as same as that inwater (�5.5 min), and was not influenced by the presenceof the chondrocytes. As shown in Fig. 4a, the wet weightof the chitosan hydrogel/cells construct decreased linearlywith the prolongation of culture time. However, the driedhydrogel weight showed no significant difference before6 d, and then decreased linearly with the prolongation ofculture time. Macroscopically, the construct gradually lostits shape and finally collapsed. In a control experiment withhydrogel only (Fig. 4b) in the same culture medium, boththe wet hydrogel weight and the dried hydrogel weightshowed no significant difference during the whole cultureperiod, which is consistent with that of Gel-I5 in PBS.These results imply that the culture medium had no signif-icant effect on the hydrogel degradation. The degradationshould be mainly brought about by incorporation of thechondrocytes. The cellular incorporation might influencethe completeness of the crosslinking network. Moreover,the viable chondrocytes might secrete bioactive substancesincluding enzymes, which may degrade the surroundinghydrogels as well.

Fig. 3. SEM images to show the internal structures of Gel-I5 in (a–c) PBS and in (d–f) 1 mg/ml lysozyme/PBS, and (g–i) Gel-I10 in 1 mg/ml lysozyme/PBSafter degradation at 37 �C for different times. (a, g), (b, h) and (c, i) are 1 d, 4 d and 18 d, respectively; (d–f) are 1 d, 4 d and 7 d, respectively.

Fig. 4. Variation of weight of (a) the cells/hydrogel construct and (b) the chitosan hydrogel only as a function of culture time.

28 Y. Hong et al. / Acta Biomaterialia 3 (2007) 23–31

3.3. In vitro growth of chondrocytes encapsulated in chitosan

hydrogel

Another important issue for the injectable hydrogel isits encapsulation property for the target cells. In mostcases, injectable hydrogels are used as scaffolds for regen-eration and repair of bone and cartilage in tissue engi-neering. Since the hydrogel microstructure and highwater content are very similar to that of the extracellularmatrix of natural cartilage, hydrogel scaffolds benefit forpreserving the prototype of chondrocytes. From theabove results, the system of 1% CML/PBS and 5 mMAPS/TMEDA was used for encapsulating chondrocytes.

The encapsulated chondrocytes showed improved viabil-ity within the hydrogel along with prolongation of theculture time initially, and then decreased from 6d(Fig. 5a). During this culture period, within the hydrogelthe DNA content, which is proportional to the total cellamount including viable and dead cells, remainedunchanged between 3 d and 6 d (p > 0.05), and thendecreased linearly and significantly (p < 0.05). This wouldmean that the increase of the cell viability at the initialculture stage is attributed to the vigorous metabolismof the chondrocytes rather than increased cell population,while the decrease of the cytoviability at the later stage iseither caused by cell death or cell loss. Cell death usually

Fig. 5. (a) Variation in optical density (l = 570 nm) (indicative of the cytoviability) with culture time. (b) Fluorescence intensity (indicative theDNA content) as a function of culture time. Hydrogels were made by mixing 1% CML/PBS and 5 mM APS/TMEDA at 37 �C. Cell seeding density was4 · 106/ml.

Fig. 6. CLSM images to show live/dead chondrocytes encapsulated in the chitosan hydrogels for (a) 1 d, (b) 3 d, (c) 6 d, (d) 9 d and (e) 12 d. Cells werestained by FDA/EB. Hydrogels were made by mixing 1% CML/PBS and 5 mM APS/TMEDA at 37 �C. Cell seeding density was 4 · 106/ml.

Y. Hong et al. / Acta Biomaterialia 3 (2007) 23–31 29

occurs inside the hydrogel because of limited exchange ofnutrition and cell metabolism products [21]. In thisexperiment, we indeed found that a few viable cellsexisted in the inside of the hydrogel, but a larger amountof cells lived on the outside of the hydrogel. Cell loss orrelease of DNA from the dead cells will of course lead todecrease of the DNA content.

The viable and dead chondrocytes were observed byCLSM as a function of culture time (Fig. 6). At 1 d(Fig. 6a), there existed a large amount of viable and roundshaped cells (green regions)1 and many dead cells (redregions), demonstrating that part of the cells died during

1 For interpretation of color in Fig. 6, the reader is referred to the webversion of this article.

the gelation procedure. The images at 3 d (Fig. 6b) and6 d (Fig. 6c) show a similar cell population as that of 1 d,except for the fewer number of dead cells. At 9 d(Fig. 6d) and 12 d (Fig. 6e), the number of viable cellswas decreased obviously. These observations are consistentwith those on cell viability and DNA content.

The morphology of chondrocytes was further observedby SEM (Fig. 7). Elliptical or round shaped chondrocyteswere uniformly distributed in the chitosan hydrogel afterin vitro culture for 3 d (Fig. 7a) and 12 d (Fig. 7c). Thecells resided in the cavities or by the edges of the cavities,and some were still capsulated within the chitosan hydro-gel. The existence of the cavities reflects the living spacespreserved by the cells. From Fig. 7b and Fig. 7d, one canestimate the cell size as �10 lm, and conclude there iseffective interaction between the cells and the hydrogel.

Fig. 7. SEM images to show morphology of the chondrocytes encapsulated in the chitosan hydrogels for (a) 3 d and (c) 12 d. (b) and (d) are highermagnifications of (a) and (c), respectively, to show single cell. Hydrogels were made by mixing 1% CML/PBS and 5 mM APS/TMEDA at 37 �C. Cellseeding density was 4 · 106/ml.

30 Y. Hong et al. / Acta Biomaterialia 3 (2007) 23–31

Particularly after being cultured for 12 d (Fig. 7d), a thinfiber-like matrix secreted by the cell bridged between thecell and the cavity walls. Comparing the cell numbers,one can conclude further that there are more cells afterbeing cultured for 3 d (Fig. 7a) than for 12 d (Fig. 7c),indicating the reduction of cell population after a longerculture period. All these results demonstrate that chon-drocytes can survive in the chitosan hydrogel and possessnormal morphology, as per that in normal cartilage, pre-dicting a potential application of the hydrogel as aninjectable scaffold. However, further optimization of thesystem is required to promote cell proliferation andECM production besides maintenance of their phenotype;for example, introduction of cell carriers is underinvestigation.

4. Conclusions

The chitosan hydrogel formed at a lower initiator con-centration, e.g., 5 mM APS/TMEDA, degrades rapidlyeither in lysozyme solution or in the case of chondrocyteencapsulation, but does not degrade in PBS solution duringan 18 d-incubation. With a higher initiator concentration,e.g., 10 mM APS/TMEDA, the hydrogel formed does notdegrade even in lysozyme solution due to the higher cross-linking degree. In vitro chondrocyte encapsulation demon-strates that the cells can survive in the chitosan hydrogeland possess normal morphology through a 12 d-cultureperiod, although the overall cell number is decreased asdetected by DNA assay. These features make the chitosanhydrogel potentially applicable as an injectable scaffold fortissue regeneration.

Acknowledgements

This study was financially supported by the Major StateBasic Research Program of China (2005CB623902), Ph.D.Programs Foundation of Ministry of Education of China(20050335035), and the National Science Fund for Distin-guished Young Scholars of China (No. 50425311).

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